We are developing techniques of blocking of MLC reactions (mixed lymphocyte culture) which will correlate with the in vivo enhancing capabilities of alloimmune or other forms of "enhancing" sera. Two major difficulties have been encountered. First, there are at least four factors present in any serum, immune or non-immune, which nonspecifically affect H3-thymidine incorporation into cultured lymphocytes. These are (1) a positive serum protein growth factor (cultures survive better in 5-10% serum than in lesser amounts), (2) a serum protein inhibitory factor (cultures survive less well in 20% or higher amounts than in 10% serum), (3) an inhibitory protein factor present in erythrocyte lysates, presumably hemoglobin, and (4) a growth factor, also present in RBC lysates, which reverses the inhibitory effect of the RBC lysate or hemoglobin, and which can be replaced by 2-mercaptoethanol. Since these factors affect the incorporation of thymidine into both mixed and unmixed control cells, stimulation indices are a better guide to transformation than the absolute level of incorporation of thymidine. SI is A x Bm over A x Am. The second problem is with the specificity of inhibition of the MLC. L anti-BN serum (serum made in Lewis rats in response to Brown Norway lymphocytes injected intravenously 4 to 6 days earlier) inhibits not only L x BNm combinations, but also L x Bum, Bu x Lm, BN x Lm, Bn x Bum. As long as one of the reactants is Lewis or Brown Norway, the MLC is inhibited. Completely different combinations are not blocked (Bu x Dam). Adsorption of the serum with BN or L cells does not remove all activity.